The present invention relates to novel, remarkably rigid hydrogels and, more particularly, to peptide-based hydrogels, to methods of generating same, to compositions containing same and to uses thereof in applications such as drug delivery, cell growth, tissue engineering, tissue regeneration, cosmetics, implantation, packaging and more.
Biological building blocks construct complex architectures and machinery through the process of molecular self-assembly. This process offers a new direction for the design and fabrication of novel materials that can be used in various applications such as microelectronics, drug delivery, and tissue engineering. Designed and well-ordered structures are formed by in vitro self-assembly of nucleic acid, phospholipid and polypeptide building blocks. Furthermore, the structural and chemical diversity of natural and nonstandard residues that could be integrated into proteins and polypeptides confers upon them some advantages over other building blocks for constructing complex architectures [Reches M. et al., Nano Lett. 4, 581 (2004)].
One example of the use of simple proteins as building blocks is the technological application of the proteinaceous supermolecular structures of amyloid fibrils for the fabrication of composite nanomaterial.
Amyloid fibrils are naturally occurring self-assembled nanostructures associated with various diseases of unrelated origin. Amyloid fibrils can serve, for example, as natural templates for the fabrication of metallic nanowires by using molecular biology tools to insert metal binding elements into the amyloid forming protein sequences.
Such an incorporation of metal binding residues into fibrous assemblies is even simpler when short peptides are used as building blocks. Like proteins and large polypeptides, short peptides, too, can self-assemble into various nanostructures such as spheres, tubes, and tapes. These self-assembled nanostructures can be designed to contain organic or inorganic binding domains and other functional groups for the purpose of novel composites fabrication [Petka, W. A. et al., Science. 281, 389 (1998)].
PCT International Patent Application Nos. PCT/IL03/01045 (WO 2004/052773) and PCT/IL2004/000012 (WO 2004/060791) disclose that a remarkably short peptide, the diphenylalanine aromatic core of the β-amyloid polypeptide, efficiently self-assembles into a novel class of peptide nanotubes. These peptide nanotubes spontaneously self-assemble in aqueous solution into individual entities with long persistence length and unique mechanical properties. These peptide nanotubes can serve, for example, as a casting mold for metal nanowires and for the fabrication of peptide-nanotube platinum-nanoparticle composites. In addition, these nanotubes can also be used in electrochemical biosensing platforms. It has been suggested that aromatic interactions may have a key role in the formation of these tubular structures as they contribute free energy of formation as well as order and directionality to the self-assembly process.
PCT International Patent Application No. PCT/IL2005/000954 and Reches and Gazit [Israel J. Chem. 45, 363-371 (2005)] disclose the assembly of similar tubular and fibrillar (amyloid-like) structures by non-charged, end-capping modified aromatic peptides and, particularly, by diphenylalanine analogs such as, for example, Boc-Phe-Phe-COOH and Fmoc-Phe-Phe-COOH peptides. Most intriguingly, it has been shown that the Fmoc-Phe-Phe peptide forms fibrillar structures that are very similar in their ultra-structure and molecular dimensions to the amyloid fibrils formed by other, much longer, polypeptides. Thus, this modified dipeptide represents the smallest structural unit that can form typical amyloid-like fibrils.
Hydrogels are networks made of water-soluble natural or synthetic polymer chains and which typically contain more than 99% water. Hydrogels are of great interest as a class of materials for tissue engineering and regeneration, as they offer three dimensional (3D) scaffolds to support the growth of cultured cells.
A variety of synthetic materials, such as, for example, poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA) and farmarate-co-ethylene glycol (P(PF-co-EG), may be used as hydrogel forming materials. These synthetic building blocks offer controllability and reproducibility, but their drawbacks lie in their production, which often involves extreme temperatures and pressures and complex techniques, as well as in the low biocompatibility of the formed hydrogel.
Another class of building blocks for hydrogel formation includes natural polymers such as agarose, collagen, fibrin, alginate, gelatin, and hyaluronic acid (HA). These polymers are appealing for medical use (e.g., medical devices or portions thereof, stents, anastomosis, adhesives etc.) due to their similarity to a natural extracellular matrix (ECM), which allows for cell adhesion while maintaining very good biocompatible and biodegradable qualities.
Protein or peptide-based scaffolds represent another very important biocompatible material that can support cell growth. Peptide-based hydrogels incorporate the advantages of both synthetic and naturally derived hydrogel-forming materials. They are easy to manufacture in large quantities and can also be easily decorated chemically and biologically. Such decoration allows the design of an ultra-structure that presents ligands, as well as other functional groups, hence promoting cell adhesion and growth [Silva et al., Science 303, 1352 (2004); Holmes et al. Proc. Natl. Acad. Sci. U.S.A. 97, 6728 (2000)].
Proteins and peptides can also form unique materials at macroscopic along with nanoscopic levels, such as nano-scale ordered hydrogels [see, for example, Holmes et al. 2000 (supra)].
Zhang et al. [J. Am. Chem. Soc. 125, 13680-13681 (2003)] have reported that supramolecular hydrogels can be formed from various Fmoc-dipeptides, at a low pH value of 3-5, and a temperature below 74° C.
The Fmoc-dipeptides practiced in this study by Zhang et al. have no aromatic substituent, apart from the Fmoc group. In the absence of aromatic substituents, the dipeptides composing the hydrogel can freely interact, via hydrogen bonding, with an external ligand. Indeed, it was reported that in the presence of an external ligand such as vancomycin, the ligand interacts with the gel through four to five hydrogen bonds, leading to its transition into a sol. Therefore, some of these hydrogels, such as those formed from Fmoc-D-Ala-D-Ala, have been shown to exhibit gel-sol transition upon binding to an external ligand such as vancomycin via a ligand-receptor interaction that can disturb the delicate balance between hydrophobic interactions and hydrogen bonds within the gel and induce a gel-sol transition.
Amino acid-derived anti-inflammatory agents have also been used to form hydrogels [Yang Z. et al., Chem. Commun. 208-209 (2004)]. In this case, a hydrogel was formed by the combination of two N-(fluorenyl methoxycarbonyl)amino acids: NPC 15199 and Fmoc-L-lysine, which belong to a novel class of anti-inflammatory agents. According to this study, NPC 15199 serves as the structural component which offers anti-inflammatory function and hence can possibly serve as a “self-delivery” system. Neither NPC 15199 nor Fmoc-L-lysine, however, can form a hydrogel independently because of their limited solubility in water. Therefore, addition of Na2CO3 to the suspension of either of the building blocks is required, forming salt type hydrogel building blocks. The pH value of the resulting hydrogel is about 9.1 and a temperature below 51° C. is required to maintain gelation.
A commercial PuraMatrix hydrogel (from BD Biosciences, http://www.bdbiosciences.com/discovery_labware/Products/tis sue_engineering/PuraM atrix/index.shtml) is also available and is used to create defined three-dimensional (3D) microenvironments for a variety of cell culture experiments. The PuraMatrix hydrogel is based on peptides that have six or more amino acid residues, for example peptides based on the (RARADADA)n motif, that have positively charged arginines and negatively charged aspartic acids [Mang S., Biotechnology Advances, 321-339 (2002)]. The peptide building blocks used form beta sheet structures in aqueous solution as they contain two distinct surfaces, one hydrophilic, the other hydrophobic. These peptides therefore form complementary ionic bonds with regular repeats on the hydrophilic surface [Zhang S. et al., Proc. Natl. Acad. Sci. USA 90, 3334-3338 (1993); Zhang S. et al., Biomaterials 16, 1385-1393 (1995)]. However, as is indicated in the manufacturer's website (supra) this hydrogel forms a soft fibrous network that exhibits a relatively weak mechanical strength, and hence its handling and use in various applications is limited. For example, it was reported that the ionic peptide [COCH3]-RADARADARADARADA-[CONH2](SEQ ID NO: 1) undergoes molecular self-assembly into nanofibers and eventually a scaffold hydrogel. Rheological analyses for this hydrogel showed an increase of scaffold rigidity as a function of nanofiber length. However, a G′ value of only about 50 Pa at 1 Hz frequency was measured for this gel [Mang S. et al. Proc. Natl. Acad. Sci. USA 102, 8414-8419 (2005)].
Hence, while the advantageous use of peptide-based hydrogels has been widely recognized, the presently known peptide-based hydrogels are characterized by relatively low rigidity, instability under certain conditions and/or the complexity of the preparation thereof.
There is thus a widely recognized need for, and it would be highly advantageous to have, novel peptide-based hydrogels and articles made therefrom, which are devoid of the above limitations.